The present invention relates to humanized antibodies derived from chimeric antibody NR-LU-13 or other antibodies having the same or similar binding specificity, fragments thereof (including, e.g., variable regions and scFv""s), conjugates (including fusion proteins) containing such humanized antibodies or fragments, and the use of such humanized antibodies or fragments in diagnostic and therapeutic pretargeting methods and compositions. The present invention also relates to the use of such humanized antibodies in conventional immunotherapeutic and immunodiagnostic methods and compositions, e.g., for tumor treatment and imaging.
A specific antibody which has been previously disclosed to be an effective targeting moiety is NR-LU-10, a murine monoclonal antibody produced against a human cancer antigen. NR-LU-10 is a nominal 150 kilodalton molecular weight murine IgG2b pancarcinoma monoclonal antibody that recognizes an approximately 40 kilodalton glycoprotein antigen expressed on most carcinomas. NR-LU-10 has been safely administered to hundreds of patients in human clinical trials. However, its disadvantage is that it is a murine derived monoclonal antibody. This is disadvantageous because immunogenicity may potentially reduce targeting efficacy if the antibody is administered repeatedly. While therapeutic efficacy may be obtained using a single administration, multiple administrative protocols are currently favored.
As a means of reducing immunogenicity of murine antibodies, various methods have been reported in the literature. Such methods include the production of chimeric antibodies which contain murine variable regions and human constant regions, the production of single chain antibodies which comprise variable binding sequences derived from murine antibodies, the production of antigen-binding fragments of murine antibodies which because of their smaller size are potentially less immunogenic, the production of human monoclonal antibodies and the production of xe2x80x9chumanizedxe2x80x9d antibodies.
Murine monoclonal antibodies may be made more human-like, e.g., by genetically recombining the nucleotide sequence encoding the murine Fv region (i.e., containing the antigen binding sites) or the complementarity determining regions thereof with nucleotide sequences encoding human constant region sequences (comprised in the Fc region of antibody). These antibodies are typically referred to as chimeric antibodies.
In this regard, a chimeric antibody derived from NR-LU-10, referred to as NR-LU-13, has previously been reported. This antibody contains the murine Fv region of NR-LU-10 and therefore comprises the same binding specificity as NR-LU-10. Thus, this chimeric antibody binds the NR-LU-10 antigen.
Humanization ideally provides an antibody that is non-immunogenic, with complete retention of the antigen-binding properties of the parent non-human antibody molecule. Non-immunogenicity allows for the administration of multiple dosages without adverse immunogenic reaction. Various methods for producing humanized antibodies have been reported in the literature. For example, humanized antibodies can potentially be produced: (a) by grafting only the non-human CDRs onto human framework and constant regions (Jones et al., Nature 321:522-25 (1986); Verhoeyen et al., Science 239:1534-1536 (1988)); or (b) by transplanting the entire non-human variable domains (to preserve ligand-binding properties) but also xe2x80x9ccloakingxe2x80x9d them with a human-like surface by replacement of exposed residues to reduce immunogenicity (also referred to as xe2x80x9cveneeredxe2x80x9d antibodies) (Padlan, Molec. Immun. 28:489-498 (1991); Padlan, Molec. Immun. 31(3):169-217 (1994)).
Retention of murine residues within human variable region framework domains reportedly helps retain proper binding function of the resultant humanized antibody. Humanized antibodies have been reported to potentially decrease or eliminate the immunogenicity of the antibody in a host recipient, thereby permitting an increase in the bioavailability and a reduction in the possibility of adverse immune reactions, thus potentially enabling multiple antibody administrations. Also, the synthesis of scFv and antibody fragments such as Fv, Fd, Fab, Fabxe2x80x2, and F(ab)xe2x80x22 fragments, derived from antibodies having a desired binding specificity comprises another known means of producing targeting moieties having lesser immunogenicity than intact antibodies. Essentially, single chain antibodies and antibody fragments because of their smaller size could be less immunogenic than intact antibodies.
It is also known that recombinant proteins, e.g., antibodies, are glycosylated differently in different host cells used for expression. Essentially, different host cells have a characteristic manner by which they glycosylate specific sites on proteins referred to as glycosylation sites or glycosylation motifs.
For example, plant cells primarily glycosylate proteins by O-linked glycosylation, whereas animal cells typically glycosylate proteins by N-linked and O-linked glycosylation. Also, the specific carbohydrates and the glycosylation pattern varies dependent upon the particular host cells.
It has been reported in the literature that oligosaccharides may be significant insofar as the targeting of proteins to specific sites. Moreover, it is also known that carbohydrates may elicit an immunogenic response. Accordingly, there is the possibility that proteins expressed in foreign host cells may elicit an immunogenic response because of carbohydrate residues which are introduced by the host cells used for expression. This is particularly problematic if the foreign host cells glycosylate very differently from humans. For example, there is the possibility that mammalian proteins expressed in plant cells may be immunogenic because plant cells glycosylate proteins very dissimilarly to mammalian cells.
Due to the difficulties related to immunogenicity of murine or chimeric antibodies that bind to the antigen bound by antibody NR-LU-13, there is a need in the art for improved compositions and methods. The present invention fulfills this need and further provides other related advantages.
It is an object of the invention to provide humanized antibodies derived from NR-LU-13 (or from other non-human antibodies which bind the antigen bound by NR-LU-13) or fragments of such humanized antibodies, which exhibit reduced immunogenicity or toxicity in humans but retain the ability to bind the NR-LU-13 antigen.
It is another object of the invention to provide conjugates containing humanized antibodies derived from NR-LU-13 or from other non-human antibodies or fragments thereof which bind the antigen bound by NR-LU-13.
It is still another object of the invention to provide improved two-step pretargeting methods wherein the improvement comprises using as the targeting moiety a humanized antibody derived from NR-LU-1 3 or from another non-human antibody or fragments thereof which bind the antigen bound by NR-LU-13.
It is another object of the invention to provide improved three-step targeting methods wherein the improvement comprises using as the targeting moiety a humanized antibody derived from NR-LU-13 or from another non-human antibody or fragments thereof which bind the antigen bound by NR-LU-13.
It is yet another object of the invention to provide compositions for treatment or diagnosis which contain conjugates comprising humanized antibodies derived from NR-LU-13 or from other non-human antibodies or fragments thereof which bind the antigen bound by NR-LU-13.
It is a more specific object of the invention to provide conjugates comprising a humanized antibody derived from NR-LU-13 or a fragment thereof capable of binding the antigen bond by NR-LU-13, directly or indirectly attached to a member of a ligand or anti-ligand partner, preferably avidin or streptavidin or a fragment or derivative thereof capable of binding biotin.
It is another object of the invention to provide a conjugate comprising a humanized antibody derived from NR-LU-13 or a fragment thereof, which binds the antigen bound by NR-LU-13, for use in a method of treating or diagnosing cancer.
It is an even more specific object of the invention to produce specific humanized variable heavy and light sequences derived from NR-LU-13 referred to herein as humanized NRX451 or fragments thereof which bind to the antigen bound by NR-LU-13.
It is another specific object of the invention to provide compositions for treating or diagnosing cancer using humanized NRX451 or fragments thereof which bind the antigen bound by NR-LU-13.
It is another object of the invention to produce antibodies, in particular, murine, chimeric or humanized antibodies which have been mutated so as to eliminate one or more potential glycosylation sites and thereby reduce immunogenicity or toxicity.
It is another object of the invention to use antibodies, preferably humanized antibodies, which have been mutated to eliminate N-linked glycosylation or modified to reduce N-linked glycosylation, in pretargeting methods and conventional antibody therapy.
Thus, the present invention provides a humanized antibody or an antigen-binding humanized antibody fragment, wherein the antibody or the antibody fragment binds specifically to the antigen bound by antibody NR-LU-13, and preferably wherein the antibody or the antibody fragment either does not possess N-linked glycosylation or its N-linked glycosylation has been modified post expression to reduce immunogenicity or toxicity. The present invention also provides a method of reducing immunogenicity or toxicity of an antibody or an antigen-binding antibody fragment of IgG class, comprising the steps of: (a) selecting a host system for the characteristic that the system does not N-link glycosylate an antibody or an antibody fragment; and (b) expressing in the host system a nucleotide sequence comprising nucleic acids (e.g., DNA or RNA or functional equivalents) encoding an IgG antibody or an antigen-binding antibody fragment. The present invention further provides a method of eliminating N-linked glycosylation in an antibody or an antigen-binding antibody fragment of IgG class to reduce immunogenicity or toxicity, comprising expressing in a host system a nucleotide sequence comprising nucleic acids (e.g., DNA or RNA or functional equivalents) encoding an IgG antibody or an antigen-binding antibody fragment, wherein the host system does not N-link glycosylate the antibody or the antibody fragment. The present invention further provides a method of modifying the N-linked glycosylation of an antibody or an antigen-binding antibody fragment of IgG class (e.g., to reduce immunogenicity or toxicity), comprising subjecting the antibody or antibody fragment to a post expression modification that modifies the N-linked glycosylation. In a preferred embodiment, antibodies or fragments of IgG. class are modified chemically to reduce immunogenicity or toxicity.
Conjugates are provided comprising a humanized antibody or antibody fragment of the present invention, attached directly or indirectly to a ligand, anti-ligand, diagnostic agent or therapeutic agent. Pharmaceutical compositions are provided comprising an antibody or antibody fragment or conjugate of the present invention, in combination with a pharmaceutically acceptable carrier or diluent. An antibody or antibody fragment or conjugate or composition of the present invention is provided for use as a diagnostic or as a medicament; for use in diagnostic or therapeutic pretargeting methods; and in methods for the diagnosis of cancer, or in methods for the treatment of cancer. In diagnostic or therapeutic methods, an antibody or antibody fragment or conjugate or composition of the present invention is administered to a warm-blooded animal (such as a human) in an amount effective for diagnosis or therapy, respectively.
These and other embodiments of the present invention will become evident upon reference to the following detailed description and attached drawings.